Cross-linked poly(trimethylene carbonate-co-L-lactide) as a biodegradable, elastomeric scaffold for vascular engineering applications

A series of copolymers of trimethylene carbonate (TMC) and l-lactide (LLA) were synthesized and evaluated as scaffolds for the production of artificial blood vessels. The polymers were end-functionalized with acrylate, cast into films, and cross-linked using UV light. The mechanical, degradation, and biocompatibility properties were evaluated. High TMC polymers showed mechanical properties comparable to human arteries (Young’s moduli of 1.2–1.8 MPa and high elasticity with repeated cycling at 10% strain). Over 84 days degradation in PBS, the modulus and material strength decreased gradually. The polymers were nontoxic and showed good cell adhesion and proliferation over 7 days using human mesenchymal stem cells. When implanted into the rat peritoneal cavity, the polymers elicited formation of tissue capsules composed of myofibroblasts, resembling immature vascular smooth muscle cells. Thus, these polymers showed properties which were tunable and favorable for vascular tissue engineering, specifically, the growth of artificial blood vessels in vivo.

Vascular remodeling in the internal mammary artery graft and association with in situ endothelin-1 and receptor expression

BACKGROUND: The vasoconstricting peptide endothelin-1 (ET-1) has been associated with atherosclerotic cardiovascular disease, vascular smooth muscle cell (VSMC) growth stimulation, and intimal thickening. ET-1 binds 2 receptor subtypes, endothelin A and B, and the ETA receptor mediates vasoconstriction and VSMC growth. This study aims to quantitatively assess arterial remodeling variables and compare them with changes in ET-1, ETA, and ETB expression in the internal mammary artery (IMA). METHODS AND RESULTS: Specimens from 55 coronary artery disease (CAD) patients (45 men, 10 women; mean age 65 years) and 14 control IMA specimens (from 7 men and 7 women; mean age 45 years) were collected. IMA cross sections were assessed by histochemical and immunohistochemical staining methods to quantify the levels of medionecrosis, fibrosis, VSMC growth, ET-1, ETA, ETB, and macrophage infiltration. The percentage area of medionecrosis in the patients was almost double that in the controls (31.85+/-14.52% versus 17.10+/-9.96%, P=0.0006). Total and type 1 collagen was significantly increased compared with controls (65.8+/-18.3% versus 33.7+/-13.7%, P=0.07, and 14.2+/-10.0% versus 4.8+/-2.8%, P=0.01, respectively). Despite ACE and/or statin therapy, ET-1 expression and cell cycling were significantly elevated in the patient IMAs relative to the controls (46.27+/-18.46 versus 8.56+/-8.42, P=0.0001, and 37.29+/-12.88 versus 11.06+/-8.18, P=0.0001, respectively). ETA and ETB staining was elevated in the patient vessels (46.88+/-11.52% versus 18.58+/-7.65%, P=0.0001, and 42.98+/-7.08% versus 34.73+/-5.20%, P=0.0067, respectively). A mild presence of macrophages was noted in all sections. CONCLUSIONS: Elevated distribution of collagen indicative of fibrosis coupled with increased cell cycling and high levels of ET-1 and ETA expression in the absence of chronic inflammation suggests altered IMA VSMC regulation is fundamental to the remodeling process.

Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production

BACKGROUND Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.

GABAB receptors are expressed in human aortic smooth muscle cells and regulate the intracellular Ca2+ concentration

The aim of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter gamma-aminobutyric acid (GABAB), in human aortic smooth muscle cells (HASMCs), and to explore if altering receptor activation modified intracellular Ca(2+) concentration ([Ca(2+)]i) of HASMCs. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABABR1 and GABABR2 in cultured HASMCs. Immunohistochemistry was used to localize the two subunits in human left anterior descending artery (LAD). The effects of the GABAB receptor agonist baclofen on [Ca(2+)]i in cultured HASMCs were demonstrated using fluo-3. Both GABABR1 and GABABR2 mRNA and protein were identified in cultured HASMCs and antibody staining was also localized to smooth muscle cells of human LAD. 100 μM baclofen caused a transient increase of [Ca(2+)]i in cultured HASMCs regardless of whether Ca(2+) was added to the medium, and the effects were inhibited by pre-treatment with CGP46381 (selective GABAB receptor antagonist), pertussis toxin (a Gi/o protein inhibitor), and U73122 (a phospholipase C blocker). GABAB receptors are expressed in HASMCs and regulate the [Ca(2+)]i via a Gi/o-coupled receptor pathway and a phospholipase C activation pathway

Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

Supernatants of acquired immunodeficiency syndrome-related Kaposi's sarcoma cells induce endothelial cell chemotaxis and invasiveness

Kaposi's sarcoma (KS) in general, and acquired immunodeficiency syndrome-related KS (AIDS-KS) in particular, is a highly invasive and intensely angiogenic neoplasm of unknown cellular origin. We have recently established AIDS-KS cells in long term culture and reported the development of KS-like lesions in nude mice inoculated with these cells. Here, we have examined the in vitro invasiveness of basement membrane by AIDS-KS cells, as well as the effect(s) of their supernatants on the migration and invasiveness of human vascular endothelial cells. AIDS-KS cells were highly invasive in the Boyden chamber invasion assay and formed invasive, branching colonies in a 3-dimensional gel (Matrigel). Normal endothelial cells form tube-like structures on Matrigel. AIDS-KS cell-conditioned media induced endothelial cells to form invasive clusters in addition to tubes. KS-cell-conditioned media, when placed in the lower compartment of the Boyden chamber, stimulated the migration of human and bovine vascular endothelial cells across filters coated with either small amounts of collagen IV (chemotaxis) or a Matrigel barrier (invasion). Basic fibroblast growth factor could also induce endothelial cell chemotaxis and invasion in these assays. However, when antibodies to basic fibroblast growth factor were used the invasive activity induced by the AIDS-KS-cell-conditioned media was only marginally inhibited, suggesting that the large quantities of basic fibroblast growth factor-like material released by the AIDS-KS cells are not the main mediators of this effect. Specific inhibitors of laminin and collagenase IV action, which represent critical determinants of basement membrane invasion, blocked the invasiveness of the AIDS-KS cell-activated endothelial cells in these assays. These data indicate that KS cells appear to be of smooth muscle origin but secrete a potent inducer of endothelial cell chemotaxis and invasiveness which could be responsible for angiogenesis and the resulting highly vascularized lesions. These assays appear to be a model to study the invasive spread and angiogenic capacity of human AIDS-related KS and should prove useful in the identification of molecular mediators and potential inhibitors of neoplastic neovascularization.

Time course analysis of hypoxia, granulation tissue and blood vessel growth, and remodeling in healing rat cutaneous incisional primary intention wounds

Hypoxia and the development and remodeling of blood vessels and connective tissue in granulation tissue that forms in a wound gap following full-thickness skin incision in the rat were examined as a function of time. A 1.5 cm-long incisional wound was created in rat groin skin and the opposed edges sutured together. Wounds were harvested between 3 days and 16 weeks and hypoxia, percent vascular volume, cell proliferation and apoptosis, α-smooth muscle actin, vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2, and transforming growth factor-β 1 expression in granulation tissue were then assessed. Hypoxia was evident between 3 and 7 days while maximal cell proliferation at 3 days (123.6 ± 22.2 cells/mm 2, p < 0.001 when compared with normal skin) preceded the peak percent vascular volume that occurred at 7 days (15.83 ± 1.10%, p < 0.001 when compared with normal skin). The peak in cell apoptosis occurred at 3 weeks (12.1 ± 1.3 cells/mm 2, p < 0.001 when compared with normal skin). Intense α-smooth muscle actin labeling in myofibroblasts was evident at 7 and 10 days. Vascular endothelial growth factor receptor-2 and vascular endothelial growth factor-A were detectable until 2 and 3 weeks, respectively, while transforming growth factor-β 1 protein was detectable in endothelial cells and myofibroblasts until 3-4 weeks and in the extracellular matrix for 16 weeks. Incisional wound granulation tissue largely developed within 3-7 days in the presence of hypoxia. Remodeling, marked by a decline in the percent vascular volume and increased cellular apoptosis, occurred largely in the absence of detectable hypoxia. The expression of vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2, and transforming growth factor-β 1 is evident prior, during, and after the peak of vascular volume reflecting multiple roles for these factors during wound healing.

The effect of cryotherapy on the vascular regeneration following closed soft tissue trauma

INTRODUCTION Icing (cryotherapy) is being widely used for the treatment of closed soft tissue trauma (CSTT), such as those resulting from sport injuries. It is believed that cryotherapy induces vasoconstriction and through this mechanism reduces inflammation [1]. However, the impact of this technique on the healing of impaired vasculature and muscle injuries following trauma remains controversial. Recent evidence suggests that the muscle regeneration is delayed after cryotherapy [2]. Consequently, we aimed to investigate the effect of cryotherapy on the vascular morphology following CSTT using an experimental model in rats by contrast-enhanced micro-CT imaging. METHODS Fifty four rats were divided into three main groups: control (no injury, n=6), sham (CSTT but no icing treatment, n=24) and icing (CSTT, treated with one session of ice block massaged directly on the injured muscle for 20 minutes, n=24). The CSTT was induced to the left thigh (Biceps Femoris) of anaesthetised rats (Male, Wistar) to create a standardized and reproducible vascular and muscle injury using an impact device [3]. Following trauma, animals were euthanized after 1, 3, 7, and 28 days healing time (n=6 for each time point). For a three-dimensional vascular morphological assessment, the blood vessels of euthanised rats were flushed with heparinised saline and then perfused with a radio-opaque contrast agent (Microfil, MV 122, Flowtech, USA) using an infusion pump. Both hind-limbs were dissected, and then the injured and non-injured limbs were imaged using a micro-CT scanner (µCT 40, Scanco Medical, Switzerland) and total volume of the perfused blood vessels (TVV) was calculated. More detailed morphological parameters such as vessel volume (VV), diameter (VD), spacing (VSp), number (VN) and connectivity (VConn) were quantified through high resolution (6 µm), micro-CT-scanned biopsy samples (diameter: 8mm) taken directly from the region of the injured muscles. The biopsies were then analysed histologically to confirm the results derived from contrast-enhanced micro-CT imaging. RESULTS AND DISCUSSION The TVV was significantly higher in the injured legs compared to the non-injured legs at day 1 and 7 in the sham group and at day 28 in both sham and icing groups. The biopsies from the injured legs of the icing group showed a significant reduction in VV, VN, VD, VConn and an increase in VSp compared to those in the sham and control groups at days 1, 3 and 7, post injury. While the injured legs of the sham group exhibited a decrease in VN and VConn 28 days post trauma, indicating a return to the original values prior to trauma, these parameters had increased in the icing group (Figure 1). Also, at day 1 post injury, VV and VD of the injured legs were significantly higher in the sham group compared to the icing group, which may be attributed to the effect of vasoconstriction induced by icing. Further histomorphological evaluation of day 1 post injury, indicated that although cryotherapy significantly reduced the injury size and influx of inflammatory cells, including macrophages and neutrophils, a delay in vascular and muscle fiber regeneration was found at later time points confirming other reports from the literature [2]. CONCLUSIONS We have demonstrated using micro-CT imaging that the vascular morphology changes after CSTT, and that its recovery is affected by therapeutic modalities such as icing. This may be useful for the development of future clinical monitoring, diagnosis and treatment of CSTT. While icing reduces the swelling after trauma, our results suggest that it may delay the recovery of the vasculature in the injured tissue.

GABAB receptors expressed in human aortic endothelial cells mediate intracellular calcium concentration regulation and endothelial nitric oxide synthase translocation

GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i) in a number of cells (e.g., retina, airway epithelium and smooth muscle), but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in cultured human aortic endothelial cells (HAECs), and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS) translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

Meta-analysis of 375,000 individuals identifies 38 susceptibility loci for migraine

Migraine is a debilitating neurological disorder affecting around 1 in 7 people worldwide, but its molecular mechanisms remain poorly understood. Some debate exists over whether migraine is a disease of vascular dysfunction, or a result of neuronal dysfunction with secondary vascular changes. Genome-wide association (GWA) studies have thus far identified 13 independent loci associated with migraine. To identify new susceptibility loci, we performed the largest genetic study of migraine to date, comprising 59,674 cases and 316,078 controls from 22 GWA studies. We identified 45 independent single nucleotide polymorphisms (SNPs) significantly associated with migraine risk (P < 5 x 10-8) that map to 38 distinct genomic loci, including 28 loci not previously reported and the first locus identified on chromosome X. Furthermore, a subset analysis for migraine without aura (MO) identified seven of the same loci as from the full sample, whereas no loci reached genome-wide significance in the migraine with aura (MA) subset. In subsequent computational analyzes, the identified loci showed enrichment for genes expressed in vascular and smooth muscle tissues, consistent with a predominant theory of migraine that highlights vascular etiologies.

Processing of polycaprolactone and polycaprolactone-based copolymers into 3D scaffolds and their cellular responses

Synthetic polymers have attracted much attention in tissue engineering due to their ability to modulate biomechanical properties. This study investigated the feasibility of processing poly(varepsilon-caprolactone) (PCL) homopolymer, PCL-poly(ethylene glycol) (PEG) diblock, and PCL-PEG-PCL triblock copolymers into three-dimensional porous scaffolds. Properties of the various polymers were investigated by dynamic thermal analysis. The scaffolds were manufactured using the desktop robot-based rapid prototyping technique. Gross morphology and internal three-dimensional structure of scaffolds were identified by scanning electron microscopy and micro-computed tomography, which showed excellent fusion at the filament junctions, high uniformity, and complete interconnectivity of pore networks. The influences of process parameters on scaffolds' morphological and mechanical characteristics were studied. Data confirmed that the process parameters directly influenced the pore size, porosity, and, consequently, the mechanical properties of the scaffolds. The in vitro cell culture study was performed to investigate the influence of polymer nature and scaffold architecture on the adhesion of the cells onto the scaffolds using rabbit smooth muscle cells. Light, scanning electron, and confocal laser microscopy showed cell adhesion, proliferation, and extracellular matrix formation on the surface as well as inside the structure of both scaffold groups. The completely interconnected and highly regular honeycomb-like pore morphology supported bridging of the pores via cell-to-cell contact as well as production of extracellular matrix at later time points. The results indicated that the incorporation of hydrophilic PEG into hydrophobic PCL enhanced the overall hydrophilicity and cell culture performance of PCL-PEG copolymer. However, the scaffold architecture did not significantly influence the cell culture performance in this study.

In vitro and in vivo analysis of co-electrospun scaffolds made of medical grade poly(epsilon-caprolactone) and porcine collagen

In this study, a nanofiber mesh made by co-electrospinning medical grade poly(epsilon-caprolactone) and collagen (mPCL/Col) was fabricated and studied. Its mechanical properties and characteristics were analyzed and compared to mPCL meshes. mPCL/Col meshes showed a reduction in strength but an increase in ductility when compared to PCL meshes. In vitro assays revealed that mPCL/Col supported the attachment and proliferation of smooth muscle cells on both sides of the mesh. In vivo studies in the corpus cavernosa of rabbits revealed that the mPCL/Col scaffold used in conjunction with autologous smooth muscle cells resulted in better integration with host tissue when compared to cell free scaffolds. On a cellular level preseeded scaffolds showed a minimized foreign body reaction.

Comparative effects of posterior eye cup tissues from Myopic and Hyperopic chick eyes on cultured scleral fibroblasts

The role of individual ocular tissues in mediating changes to the sclera during myopia development is unclear. The aim of this study was to examine the effects of retina, RPE and choroidal tissues from myopic and hyperopic chick eyes on the DNA and glycosaminoglycan (GAG) content in cultures of chick scleral fibroblasts. Primary cultures of fibroblastic cells expressing vimentin and -smooth muscle actin were established in serum-supplemented growth medium from 8-day-old normal chick sclera. The fibroblasts were subsequently co-cultured with posterior eye cup tissue (full thickness containing retina, RPE and choroid) obtained from untreated eyes and eyes wearing translucent diffusers (form-deprivation myopia, FDM) or -15D lenses (lens-induced myopia, LIM) for 3 days (post hatch day 5 to 8) (n=6 per treatment group). The effect of tissues (full thickness and individual retina, RPE, and choroid layers) from -15D (LIM) versus +15D (lens-induced hyperopia, LIH) treated eyes was also determined. Refraction changes in the direction predicted by the visual treatments were confirmed by retinoscopy prior to tissue collection. Glycosaminoglycan (GAG) and DNA content of the scleral fibroblast cultures were measured using GAG and PicoGreen assays. There was no significant difference in the effect of full thickness tissue from either FDM or LIM treated eyes on DNA and GAG content of scleral fibroblasts (DNA 8.9±2.6 µg and 8.4±1.1 µg, p=0.12; GAG 11.2±0.6 µg and 10.1±1.0 µg, p=0.34). Retina from LIM eyes did not alter fibroblast DNA or GAG content compared to retina from LIH eyes (DNA 27.2±1.7 µg versus 23.2±1.5 µg, p=0.21; GAG 28.1±1.7 µg versus. 28.7±1.2 µg, p=0.46). Similarly, the choroid from LIH and LIM eyes did not produce a differential effect on DNA content (DNA, LIM 46.9±6.4 versus LIH 51.5±4.7 µg, p=0.31), whereas GAG content was higher for cells in co-culture with choroid from LIH eyes (GAG 32.5±0.7 µg versus 18.9±1.2 µg, F1,6=9.210, p=0.0002). In contrast, fibroblast DNA was greater in co-culture with RPE from LIM eyes than the empty basket and DNA content less for co-culture with RPE from LIH eyes (LIM: 72.4±6.3 µg versus Empty basket: 46.03±1.0 µg; F1,6=69.99, p=0.0005 and LIH: 27.9±2.3 µg versus empty basket: 46.03±1.0 µg; p=0.0004). GAG content was higher with RPE from LIH eyes (LIH: 33.7±1.9 µg versus empty basket: 29.5±0.8 µg, F1,6=13.99, p=0.010) and lower with RPE from LIM eyes (LIM: 27.7±0.9 µg versus empty basket: 29.5±0.8 µg, p=0.021). GAG content of cells in co-culture with choroid from LIH eyes was higher compared to co-culture with choroid from LIM eyes (32.5±0.7 µg versus 18.9±1.2 µg respectively, F1,6=9.210, p=0.0002). In conclusion, these experiments provide evidence for a directional growth signal that is present (and remains) in the ex-vivo RPE, but that does not remain in the ex-vivo retina. The identity of this factor(s) that can modify scleral cell DNA and GAG content requires further research.

A population genomics overview of the neuronal nitric oxide synthase (nNOS) gene and its relationship to migraine susceptibility

The ubiquitous chemical messenger molecule nitric oxide (NO) has been implicated in a diverse range of biological activities including neurotransmission, smooth muscle motility and mediation of nociception. Endogenous synthesis of NO by the neuronal isoform of the nitric oxide synthase gene family has an essential role within the central and peripheral nervous systems in addition to the autonomic innervation of cerebral blood vessels. To investigate the potential role of NO and more specifically the neuronal nitric oxide synthase (nNOS) gene in migraine susceptibility, we investigated two microsatellite repeat variants residing within the 5′ and 3′ regions of the nNOS gene. Population genomic evaluation of the two nNOS repeat variants indicated significant linkage disequilibrium between the two loci. Z-DNA conformational sequence structures within the 5′ region of the nNOS gene have the potential to enhance or repress gene promoter activity. We suggest that genetic analysis of this 5′ repeat variant is the more functional variant expressing gene wide information that could affect endogenous NO synthesis and potentially result in diseased states. However, no association with migraine (with or without aura) was seen in our extensive case-control cohort (n = 579 affected with matched controls), when both the 5′ and 3′ genetic variants were investigated.